The overall objective is elucidation of the processes of iodothyronine biosynthesis on the molecular level by multidisciplinary approaches. Emphasis of the proposed research is centered on an organic chemical approach using regio- and stereo-specifically labeled substrates (3H, 14C, 2H, 13C and 18O at positions on the side chain and ring of tyrosine and its derivatives), 18O2 and (18O) water to delineate the precise molecular mechanism(s) of thyroid hormone biosynthesis. Our immediate objectives include: (1) determination of the fates of hydrogen, carbon, oxygen and nitrogen of N-acetyliiodotyrosine in the coupling process, (2) identification of "the lost side-chain" of the coupling, (3) elucidation of the kinetic mechanism and determination of the stoichiometric involvement of oxygen in the coupling reaction, (4) measurement of relative rates of T4, T3, rT3 formation in the model system, (5) attestation of our hypothesis of the push-pull lyase reaction mechanism involving oxygen, (6) formation of polyiodophenoxyiodothyronines by extended coupling reactions and assessement of the biological activity of these new compounds, and (7) assessement of the catalysis by peroxydase of the coupling reaction. The methods of approach include synthesis of regiospecifically labeled N-acetyliodotyrosines and peptides containing DIT and/or MIT; incubation in a well-defined inorganic medium (no iodoproteins or enzymes); kinetic analysis using an oxygen meter and volumetric, spectroscopic, and radioisotope techniques; product identification by nuclear magnetic resonance, high pressure liquid chromatography and other organic chemical techniques; and three-dimensional structure determination by X-ray crystallography.